CYCLIC AMP-MEDIATED RELAXATION OF AIRWAY SMOOTH MUSCLE

循环放大器介导的气道平滑肌松弛

• 批准号: 3350145
• 负责人: PRIMAL DE LANEROLLE
• 金额: $ 8.79万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-30 至 1988-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3350145
• 关键词: calcium metabolism; cholinergic agents; cyclic AMP; gel electrophoresis; histamine; hybrid antibody; muscle metabolism; muscle pharmacology; muscle relaxation; muscle tension; phosphorylation; protein kinase; radiotracer; respiratory muscles; smooth muscle; trachea

Airways obstruction is the sine qua non of asthma. Although excess mucus secretions contribute to this phenomenon, the primary cause is a decrease in airway caliber due to airway smooth muscle contraction. An increase in cellular cyclic AMP levels has been associated with the relaxation of airway smooth muscle. There are, at present, two mutually non-exclusive hypotheses for cyclic AMP-mediated relaxation. One hypothesis asserts that relaxation is mediated through the phosphorylation of myosin light chain kinase (MLCK) by cyclic AMP-dependent protein kinase; the other asserts that relaxation results from a decrease in intracellular calcium following a rise in cyclic AMP levels. We propose to use a multidisciplinary approach to determine if either, or both, of these mechanisms contribute to cyclic AMP-mediated relaxation. First, the role of cyclic AMP-mediated phosphorylation of MLCK in canine tracheal smooth muscle (canine TSM) relaxation will be studied by determining (a) the temporal relationship between MLCK phosphorylation and relaxation, (b) the sites that are phosphorylated and (c) the effect of phosphorylation on MLCK activity. Second, the interaction between MLCK phosphorylation and cytoplasmic calcium during relaxation of skinned and intact canine TSM will be studied. In these experiments, the cytoplasmic calcium concentration will be manipulated and antibodies that inhibit MLCK phosphorylation will be used to study how these processes affect each other. Finally, changes in cell calcium in response to pharmacological stimulation of intact canine TSM will be quantitated. The Ca2+ measurements will then be correlated with changes in cyclic AMP levels, MLCK phosphorylation, myosin dephosphorylation and relaxation of canine TSM. These experiments utilize physiological, pharmacological, biochemical and immunological methods. Such an approach should provide insights into the roles of two important cellular messengers (calcium and cyclic AMP) and two important enzymes (myosin and myosin light chain kinase) in regulating the contractile properties of airway smooth muscle. These studies should also indicate avenues for pharmacological manipulation of airway caliber.

气道阻塞是哮喘的必要条件。 虽然过多的粘液 分泌物有助于这种现象，主要原因是减少 由于气道平滑肌收缩导致气道口径变小。 细胞内cAMP水平的增加与细胞内cAMP水平的升高有关。 松弛气道平滑肌。 目前，两个相互 环AMP介导的松弛的非排他性假设。 一 这一假说认为，松弛是通过磷酸化介导的， 肌球蛋白轻链激酶（MLCK）通过环腺苷酸依赖性蛋白激酶（cAMP-dependent protein kinase）; 另一种认为松弛是由于细胞内 环磷酸腺苷水平上升后的钙。 我们建议使用 多学科方法来确定其中之一或两者 这种机制有助于环AMP介导的松弛。 首先，在犬中，环AMP介导的MLCK磷酸化的作用 气管平滑肌（犬TSM）松弛将通过以下方法进行研究： 确定（a）MLCK磷酸化和 松弛，（B）磷酸化的位点和（c） 磷酸化对MLCK活性的影响 二、MLCK之间的相互作用 磷酸化和细胞质钙在松弛皮肤和 将研究完整的犬TSM。 在这些实验中， 钙浓度将被操纵，并且抑制MLCK的抗体 磷酸化将被用来研究这些过程如何影响每一个 其他. 最后，细胞钙的变化，以响应药理学 对完整犬TSM的刺激进行定量。 的ca2 + 然后将测量结果与环AMP水平的变化相关联， MLCK磷酸化、肌球蛋白去磷酸化和犬TSM的松弛。 这些实验利用生理学、药理学、生物化学和生物化学方法。 免疫学方法。 这种方法应能使人们深入了解 两个重要的细胞信使（钙和环AMP）和两个 重要的酶（肌球蛋白和肌球蛋白轻链激酶）在调节 气道平滑肌的收缩特性。 这些研究还应 指出了药理学操纵气道口径的途径。